The present invention relates to arylsulfonylamino hydroxamic acid derivatives which are inhibitors of matrix metalloproteinases or the production of tumor necrosis factor (hereinafter also referred to as TNF) and as such are useful in the treatment of a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis bullosa, scleritis and other diseases characterized by matrix metalloproteinase activity, AIDS, sepsis, septic shock and other diseases involving the production of TNF.
This invention also relates to a method of using such compounds in the treatment of the above diseases in mammals, especially humans, and to the pharmaceutical compositions useful therefor.
There are a number of enzymes which effect the breakdown of structural proteins and which are structurally related metalloproteases. Matrix-degrading metalloproteinases, such as gelatinase, stromelysin and collagenase, are involved in tissue matrix degradation (e.g. collagen collapse) and have been implicated in many pathological conditions involving abnormal connective tissue and basement membrane matrix metabolism, such as arthritis (e.g. osteoarthritis and rheumatoid arthritis), tissue ulceration (e.g. corneal, epidermal and gastric ulceration), abnormal wound healing, periodontal disease, bone disease (e.g. Paget""s disease and osteoporosis), tumor metastasis or invasion, as well as HIV-infection (J. Leuk. Biol., 52 (2): 244-248, 1992).
Tumor necrosis factor is recognized to be involved in many infectious and auto-immune diseases (W. Friers, FEBS Letters, 1991, 285, 199). Furthermore, it has been shown that TNF is the prime mediator of the inflammatory response seen in sepsis and septic shock (C.E. Spooner et al., Clinical Immunology and Immunopathology, 1992, 62 S11).

or the pharmaceutically acceptable salts thereof, wherein
n is 1 to 6;
X is hydroxy, (C1-C6)alkoxy or NR1R2 wherein R1 and R2 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, piperidyl, (C1-C6)alkylpiperidyl, (C8-C10)arylpiperidyl, (C5-C9)heteroarylpiperidyl, (C6-C10)aryl(C1-C6)alkylpiperidyl, (C5-C9)heteroaryl,(C6-C10)aryl(C1-C6)alykyl,(C5-C9)heteroaryl(C1-C6)alkyl,(C6-C10)aryl(C6-C10)aryl, (C5-C10)aryl(C6-C10)aryl(C1-C5)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(C1-C6)alkyl, R5(C2-C5)alkyl, (C1-C5)alkyl(CHR5)(C1-C5)alkyl wherein R5 is hydroxy, (C1-C6)acyloxy, (C1-C6)alkoxy, piperazino, (C1-C6)acylamino, (C5-C10)aryithio, (C1-C5)alkylsulfinyl, (C5-C10)arylsulfinyl, (C1-C8)alkylsulfoxyl, (C6-C10)arylsulfoxyl, amino, (C1-C5)alkylamino,((C1-C5)alkyl)2amino,(C1-C5)acylpiperazino, (C1-C6)aklylpiperazino, (C6-C10)aryl(C1-C5)alkylpiperazino, (C5-C9)heteroaryl(C1-C6)alkylpiperazino, morpholino, thiomorpholino, piperidino or pyrrolidino;R5(C1-C6)alkyl,(C1-C5)alkyl(CHR5)(C1-C6)alkyl wherein R5 is piperidyl, (C1-C6)alkylpiperidyl, (C6-C10)arylpiperidyl, (C6-C10)aryl(C1-C6)alkylpiperidyl, (C5-C9)heteroarylpiperidyl or (C5-C9)heteroaryl(C1-C6)alkylpiperidyl; and CH(R7)COR8 wherein R7 is hydrogen, (C1-C5)alkyl, (C5-C10)aryl(C1-C5)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl, (C1-C6)alkylthio (C1-C5)alkyl, (C5-C10)arylthio(C1-C5)alkyl, (C1-C6)alkylsulfinyl(C1-C6)alkyl, (C5-C10)arylsulfinyl(C1-C6)alkyl, (C1-C5)alkylsulfonyl(C1-C6)alkyl, (C6-C10)arylsulfonyl (C1-C6)alkyl, hydroxy(C1-C6)alkyl, amino(C1-C6)alkyl, (C1-C6)alkylamino(C1-C6)alkyl, ((C1-C5)alkylamino)2(C1-C6)alkyl,R9R10NCO(C1-C6)alkyl or R9OCO(C1-C5)alkyl wherein R9 and R10 are each independently selected from the group consisting of hydrogen, (C1-C5)alkyl, (C5-C10)aryl(C1-C5)alkyl and (C5-C9)heteroaryl(C1-C6)alkyl; and R8 is R11O or R11R12N wherein R11 and R12 are each independently selected from the group consisting of hydrogen, (C1-C5)alkyl, (C5-C10)aryl(C1-C6alkyl and (C5-C9)heteroaryl(C1-C6)alkyl;
or R1 and R2, or R9 and R10, or R11 and R12 may be taken together to form an azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, indolinyl, isoindolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, (C1-C6)acylpiperazinyl, (C1-C5alkylpiperazinyl, (C5-C10)arylpiperazinyl, (C5-C9)heterorarylpiperazinyl or a bridged diazabicycloalkyl ring selected from the group consisting of 
wherein r is 1, 2 or 3;
m is 1 or 2;
p is 0 or 1; and
Q is hydrogen, (C1-C3)alkyl or (C1-C6)acyl;
R3 and R4 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, trifluoromethyl, trifluoromethyl(C1-C6)alkyl, C1-C6)alkyl (difluoromethylene), (C1-C3)alkyl(difluoromethylene)(C1-C3)alkyl, (C6-C10)aryl, (C5-C9)heteroaryl, (C6-C10)aryl(C1-C5)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl, (C6-C10)aryl (C5-C10)aryl, (C6-C10)aryl(C5-C10)aryl(C1-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl (C1-C5)alkyl, hydroxy(C1-C6)alkyl, (C1-C6)acyloxy(C1-C5)alkyl, (C1-C5)alkoxy(C1-C5)alkyl, piperazinyl(C1-C6)alkyl,(C1-C6)acylamino(C1-C5)alkyl,piperidyl,(C1-C6)alkylpiperidyl, (C5-C10)aryl(C1-C6)alkoxy(C1-C6)alkyl, (C5-C9)heteroaryl(C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)alkylthio(C1-C5)alkyl,(C5-C10)arylthio(C1-C6)alkyl,(C1-C6)alkylsulfinyl(C1-C6)alkyl, (C6-C10)arylsulfinyl(C1-C5)alkyl, (C1-C6)alkylsulfonyl(C1-C6)alkyl, (C6-C10)arylsulfonyl (C1-C6)alkyl, amino(C1-C6)alkyl, (C1-C6)alkylamino(C1-C6)alkyl, ((C1-C5)alkylamino)2(C1-C5)alkyl, R13CO(C1-C5)alkyl wherein R13 is R20O or R20R21N wherein R20 and R21 are each independently selected from the group consisting of hydrogen, (C1-C5)alkyl, (C6-C10)aryl(C1-C6)alkyl or (C5-C9)heteroaryl(C1-C5)alkyl; or R14(C1-C5)alkyl wherein R14 is (C1-C6)acylpiperazino, (C5-C10)arylpiperazino, (C5-C9)heteroarylpiperazino, (C1-C6)alkylpiperazino, morpholino, thiomorpholino, piperidino, pyrrolidino, piperidyl, (C1-C6)alkylpiperidyl, (C5-C9)heteroaryl(C1-C6)alkylpiperidyl or (C1-C6)acylpiperidyl;
or R3 and R4, or R20 and R21 may be taken together to form a (C3-C6)cycloalkyl, oxacyclohexyl, thiocyclohexyl, indanyl or tetralinyl ring or a group of the formula 
wherein R15 is hydrogen, (C1-C6)acyl, (C1-C6alkyl, (C6-C10)aryl(C1-C6)alkyl, (C5-C9)heteroary(C1-C6)alkyl or (C1-C6)alkylsulfonyl; and
Ar is (C6-C10)aryl, (C5-C9)heteroaryl, (C1-C6)alkyl(C6-C10)aryl, (C1-C5)alkoxy (C6-C10)aryl, ((C1-C6)alkoxy)2(C6-C10)aryl, (C5-C10)aryloxy(C6-C10)aryl, (C5-C9)heteroaryloxy(C6-C10aaryl, (C1-C6)alkyl(C5-C9)heteroaryl, (C1-C6)alkoxy (C5-C9)heteroaryl, ((C1-C5)alkoxy)2(C5-C9)heteroaryl, (C6-C10)aryloxy(C5-C9)heteroaryl, (C5-C9)heteroaryloxy(C5-C9)heteroaryl;
with the proviso that when either R1 or R2 is CH(R7)COR8 wherein R7 and R8 are as defined above, the other of R1 or R2 is hydrogen, (C1-C6)alkyl or benzyl.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties or combinations thereof.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, includes O-alkyl groups wherein xe2x80x9calkylxe2x80x9d is defined above.
The term xe2x80x9carylxe2x80x9d, as used herein, unless other wise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, optionally substituted by 1 to 3 substituents selected from the group consisting of fluoro, chloro, trifluoromethyl, (C1-C6)alkocxy, (C6-C10)aryloxy, trifluoromethoxy, difluoromethoxy and (C1-C6)alkyl.
The term xe2x80x9cheteroarylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic heterocyclic compound by removal of one hydrogen, such as pyridyl, furyl, pyroyl, thienyl, isothiazolyl, imidazolyl, benzimidazolyl, tetrazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, isobenzofuryl, benzothienyl, pyrazolyl, indolyl, isoindolyl, purinyl, carbazolyl, isoxazolyl, thiazolyl, oxazolyl, benzthiazolyl or benzoxazolyl, optionally substituted by 1 to 2 substituents selected from the group consisting of fluoro, chloro, trifluoromethyl, (C1-C6)alkoxy, (C6-C10)aryloxy, trifluoromethoxy, difluoromethoxy and (C1-C6)alkyl.
The term xe2x80x9cacylxe2x80x9d, as used herein, unless otherwise indicated, includes a radical of the general formula RCO wherein R is alkyl, alkoxy, aryl, arylalkyl or arylalkyloxy and the terms xe2x80x9calkylxe2x80x9d or xe2x80x9carylxe2x80x9d are as defined above.
The term xe2x80x9cacyloxyxe2x80x9d, as used herein, includes O-acyl groups wherein xe2x80x9cacylxe2x80x9d is defined above.
The compound of formula I may have chiral centers and therefore exist in different enantiomeric forms. This invention relates to all optical isomers and steroisomers of the compounds of formula I and mixtures thereof.
Preferred compounds of formula I include those wherein n is 2.
Other preferred compounds of formula I include those wherein Ar is 4-methoxyphenyl or 4-phenoxyphenyl.
Other preferred compounds of formula I include those wherein either R3 or R4 is not hydrogen.
Other preferred compounds of formula I include those wherein n is 1 and either R1 or R2 is hydrogen.
Other preferred compounds of formula I includes those wherein X is hydroxy, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and either R3 or R4 is not hydrogen.
Other preferred compounds of formula I include those wherein X is alkoky, Ar is 4-methoxyphenyl or 4-phenoxyphenyl and either R3 or R4 is not hydrogen.
Other preferred compounds of formula I include those wherein Ar is 4-methoxyphenyl or 4-phenoxyphenyl and R3 and R4 are taken together to form (C3-C6)cycloalkanyl, oxacyclohexanyl, thiocyclohexanyl, indanyl or a group of the formula 
wherein R15 is (C1-C6)acyl, (C1-C6)alkyl, (C5-C10)aryl(C1-C6)alkyl, (C5-C9)heteroaryl(C1-C6)alkyl or (C1-C6)alkylsulfonyl.
More preferred compounds of formula I are those wherein n is 2, Ar is 4-methoxyphenyl or 4phenoxyphenyl, R1 and R2 are taken together to form piperazinyl, (C1-C6)alkylpiperazinyl, (C6-C10)aryl piperazinyl or (C5-C9)heteroaryl(C1-C6)alkylpiperazinyl, and either R3 or R4 is not hydrogen or both R3 and R4 are not hydrogen.
More preferred compounds of formula I are those wherein n is 2, Ar is 4-methoxyphenyl or 4-phenoxyphenyl, R1 is hydrogen or (C1-C6)alkyl, R2 is 2-pyridylmethyl, 3-pyridylmethyl or 4-pyridylmethyl, and either R3 or R4 is not hydrogen or both R3 and R4 are not hydrogen.
More preferred compounds of formula I are those wherein n is 1, Ar is 4-methoxyphenyl or 4phenoxyphenyl, R1 is hydrogen, R2 is 2-pyridylmethyl, 3-pyridylmethyl or 4-pyridylmethyl, and either R3 or R4 is not hydrogen or both R3 and R4 are not hydrogen.
More preferred compounds of formula I are those wherein n is 2, Ar is 4-methyoxyphenyl, R1 is hydrogen or (C1-C6)alkyl and R2 is R5 (C2-C6)alkyl wherein R5 is morpholino, thiomorpholino, piperidino, pyrrolidino, (C1-C6)acylpiperazino, (C1-C6)akylpiperazino, (C6-C10)arylpiperazino, (C5-C9)heteroarylpiperazino, (C6-C10aryl (C1-C6)alkylpiperazino or (C5-C9)heteroaryl(C1-C6)alkylpiperazino and either R3 or R4 is not hydrogen or both R3 and R4 are not hydrogen.
More preferred compounds of formula I are those wherein n is 1, Ar is 4-methoxyphenyl or 4-phenoxyphenyl, R1 is hydrogen, R2 is R5 (C2-C6)alkyl wherein R5 is morpholino, thiomorpholino, piperidino, pyrrolidino, (C1-C6)acylpiperazino, (C1-C6)akylpiperazino, (C5-C10)arylpiperazino, (C5-C9)heteroarylpiperazino, (C6-C10)aryl (C1-C6)alkylpiperazino or (C5-C9)heteroaryl(C1-C5)alkylpiperazino and either R3 or R4 is not hydrogen or both R3 and R4 are not hydrogen.
Specific preferred compounds of formula I include the following:
2-(R)-N-Hydroxy-2-[(4-methoxybenzenesulfonyl)(3-morpholin-4-yl-3-oxopropyl)amino]-3methylbutyramide;
2-(R)-2-[(2-Benzylcarbamoylethyl)(4-methoxybenzenesulfonyl)amino]-N-hydroxy-3-methylbutyramide;
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)(2-[(pyridin-3-ylmethyl)-carbamoyl]ethyl)amino)-3-methylbutyramide;
2-(R)-N-Hydroxy-2([4-methoxybenzenesulfonly][2-(methylpyridin-3-ylmethylcarbamoyl)ethyl]amino)-3-methylbutyramide;
4-(3-[1-(R)-1-Hydroxycarbamoyl-2-methylpropyl)(4-methoxybenzene-sulfonyl)amino]propionyl)piperazine-1-carboxylic acid, tert-butyl ester;
2-(R)-N-Hydroxy-2-[(4-methoxybenzenesulfonyl)(3-oxo-3-piperazin-1-ylpropyl)amino)-3-methylbutyramide hydrochloride;
2-(R)-2-[(Benzylcarbamoylmethyl)(4-methoxybenzenesulfonyl)amino]N-hydroxy-3-methylbutyramide;
2-(R)-N-Hydroxy-2-([4methoxybenzenesulfonyl]-[(2-morpholin-4-ylethyl-carbamoyl)methyl]amino)-3-methylbutyramide; and
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)([(pyridin-3-ylmethyl)carbamoyl]methyl)amino)-3-methylbutyramide.
Other specific compounds of formula I include the following:
2-(R)-3,3,3-Trifluoro-N-hydroxy-2-[(methoxybenzenesulfonyl)(3-morpholin-4-yl-3-oxopropyl)amino]propionamide;
2-(R)-N-Hydroxy-2-((4-phenoxybenzenesulfonyl)[2-(methylpyridin-4-ylmethylcarbamoyl)ether]amino)-3-methylbutyramide;
4-[4-Methoxybenzenesulfonyl)(3-morpholin-4-yl-3-oxopropyl)amino]-1-methylpiperidene-4-carboxylic acid hydroxyamide;
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)-[3-(4-methylpiperazin-2-yl)-3-oxopropyl]amino)-3-methylbutyramide;
2-(R)-2-[(2-Carboxyethyl)(4-methoxybenzenesulfonyl)amino]-N-hydroxy-3-methylbutyramide;
[(2-Carboxyethyl)(3,4-dimethoxybenzenesulfonyl)amino]-N-hydroxy-acetamide
2-(R)-2[(2-Carbamoylethyl)(4-methoxybenzenesulfonyl)amino]-N-hydroxy-3-methylbutyramide
2-(R),3-(R)-3,N-Dihydroxy-2-[(4-methoxybenzenesulfonyl)(3-oxo-3-piperidin-1-ylpropyl)amino]-butyramide
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)[3-(methylpyridin-3-ylmethylcarbamoyl)propyl]amino)-3-methylbutyramide
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)[2-(methylcarboxymethylcarbamoyl)ethyl]amino)-3-methylbutyramide
2-(R)-N-Hydroxy-2-((4-methoxybenzenesulfonyl)-[(1-methylpiperidin-4-ylcarbamoyl)methyl]amino)-3-methylbutyramidecyclo
2-(R)-2-Cyclohexyl-N-hydroxy-2-((4-methoxybenzenesulfonyl)-[3-(4-methylpiperazin-1-yl)-3-oxopropyl]amino)-acetamide
2-(R)-N-Hydroxy-2-[(methoxybenzenesulfonyl)(3-morpholin-4-yl-3-oxopropyl)amino]-4-(morpholin-4-yl)butyramide.
The present invention also relates to a pharmaceutical composition for (a) the treatment of a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis bullosa, scleritis and other diseases characterized by matrix metalloproteinase activity, AIDS, sepsis, septic shock and other diseases involving the production of tumor necrosis factor (TNF) or (b) the inhibition of matrix metalloproteinases or the production of tumor necrosis factor (TNF) in a mammal, including a human, comprising an amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof, effective in such treatments and a pharmaceutically acceptable carrier.
The present invention also relates to a method for the inhibition of (a) matrix metalloproteinases or (b) the production of tumor necrosis factor (TNF) in a mammal, including a human, comprising administering to said mammal an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.
The present invention also relates to a method for treating a condition selected from the group consisting of arthritis, cancer, tissue ulceration, restenosis, periodontal disease, epidermolysis, bullosa, scleritis and other diseases characterized by matrix metalloproteinase activity, AIDS, sepsis, septic shock and other diseases involving the production of tumor necrosis factor (TNF) in a mammal, including a human, comprising administering to said mammal an amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof, effective in treating such a condition.
The following reaction Schemes illustrate the preparation of the compounds of the present invention. Unless otherwise indicated R1, R2, R3, R4, n and Ar in the reaction Schemes and the discussion that follow are defined as above. 
In reaction 1 of Scheme 1, the amino acid compound of formula VII, wherein R15 is (C1xe2x80x94C6)alkyl, benzyl, allyl or tert-butyl, is converted to the corresponding compound of formula VI by reacting VII with a reactive functional derivative of an arylsulfonic acid compound, such as an arylsulfonyl chloride, in the presence of a base, such as triethylamine, and a polar solvent, such as tetrahydrofuran, dioxane, water or acetonitrile, preferably a mixture of dioxane and water. The reaction mixture is stirred, at room temperature, for a time period between about 10 minutes to about 24 hours, preferably about 60 minutes.
In reaction 2 of Scheme 1, the arylsulfonyl amino compound of formula VI, wherein R18 is (C1xe2x80x94C6)alkyl, benzyl, allyl or tert-butyl, is converted to the corresponding compound of formula V, wherein n is 1, 3, 4, 5 or 6, by reacting VI with a reactive derivative of an alcohol of the formula. 
such as the chloride, bromide or iodide derivative, preferably the bromide derivative, wherein the R17 protecting group is (C1xe2x80x94C6)alkyl, benzyl, allyl or tert-butyl, in the presence of a base such as potassium carbonate or sodium hydride, preferably sodium hydride, and a polar solvent, such as dimethylformamide. The reaction mixture is stirred, at room temperature, for a time period between about 60 minutes to about 48 hours, preferably about 18 hours. The R17 protecting group is chosen such that it may be selectively removed in the presence of and without loss of the R16 protecting group, therefore, R17 cannot be the same as R16. Removal of the R17 protecting group from the compound of formula V to give the corresponding carboxylic acid of formula IV, in reaction 3 of Scheme 1, is carried out under conditions appropriate for that particular R17 protecting group in use which will not affect the R16 protecting group. Such conditions include; (a) saponification where R17 is (C1xe2x80x94C6)alkyl and R16 is tert-butyl, (b) hydrogenolysis where R17 is benzyl and R16 is tert-butyl or (C1xe2x80x94C6)alkyl, (c) treatment with a strong acid such as trifluoroacetic acid or hydrochloric acid where R17 is tert-butyl and R16 is (C1xe2x80x94C6)alkyl, benzyl or allyl, or (d) treatment with tributyltinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride where R17 is allyl and R16 is (C1xe2x80x94C6)alkyl, benzyl of tert-butyl.
In reaction 4 of Scheme 1, the carboxylic acid of formula IV is condensed with an amine, R1R2NH, or the salt thereof, to give the corresponding amide compound of formula III. The formation of amides from primary or secondary amines or ammonia and carboxylic acids is achieved by conversion of the carboxylic acid to an activated functional derivative which subsequently undergoes reaction with a primary or secondary amine or ammonia to form the amide. The activated functional derivative may be isolated prior to reaction with the primary or secondary amine or ammonia. Alternatively, the carboxylic acid may be treated with oxalyl chloride or thionyl chloride, neat or in an inert solvent, such as chloroform, at a temperature between about 25xc2x0 C. to about 80xc2x0 C., preferably about 50xc2x0 C., to give the corresponding acid chloride functional derivative. The inert solvent and any remaining oxalyl chloride or thionyl chloride is then removed by evaporation under vacuum. The remaining acid chloride functional derivative is then reacted with the primary or secondary amine or ammonia in an inert solvent, such as methylene chloride, to form the amide. The preferred method for the condenation of the carboxylic acid of formula IV with an amine to provide the corresponding amide compound of formula III is the treatment of IV with (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate in the presence of a base, such as triethylamine, to provide the benzotriazol-1-oxy ester in situ which, in turn, reacts with the amine, R1R2N, in an inert solvent, such as methylene chloride, at room temperature to give the amide compound of formula III.
Removal of the R16 protecting group from the compound of formula III to give the corresponding carboxylic acid of formula II, in reaction 5 of Scheme 1, is carried out under conditions appropriate for the particular R16 protecting group in use. Such conditions include; (a) saponification where R16 is lower alkyl, (b) hydrogenolysis where R16 is benzyl, (c) treatment with a strong acid, such as trifluoroacetic acid or hydrochloric acid, where R16 is tert-butyl, or (d) treatment with tributyltinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride where R16 is allyl.
In reaction 6 of Scheme 1, the carboxylic acid compound of formula II is converted to the hydroxamic acid compound of formula I by treating II with 1-(3-dimethylaminopropyl)3-ethylcarbodiimide and 1-hydroxybenztriazole in a polar solvent, such as dimethylformamide, followed by the action of hydroxylamine to the reaction mixture after a time period between about 15 minutes to about 1 hour, preferably about 30 minutes. The hydroxylamine is preferably generated in situ from a salt form, such as hydroxylamine hydrochloride, in the presence of a base, such as N-methylmorpholine. Alternatively, a protected derivative of hydroxylamine or its salt form, where the hydroxyl group is protected as a tert-butyl, benzyl or allyl ether, may be used in the presence of (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorphosphate and a base, such as N-methylmorpholine. Removal of the hydroxylamine protecting group is carried out by hydrogenolysis for a benzyl protecting group or treatment with a strong acid, such as trifluoroacetic acid, for a tert-butyl protecting group. The allyl protecting group may be removed by treatment with tributyltinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride. N,O-bis(4-methoxybenzyl)hydroxylamine may also be used as the protected hydroxylamine derivative where deprotection is achieved using a mixture of methanesulfonic acid and trifluoroacetic acid.
In reaction 1 of Scheme 2, the arylsulfonylamino compound of formula VI, wherein R16 is (C1xe2x80x94C6)alkyl, benzyl or tert-butyl, is converted to the corresponding compound of formula VIII, wherein R18 is 2-propenyl or 3-butenyl, by reacting IX with a reactive functional derivative, such as the halide, preferably the iodide derivative, of 2-propen-1-ol when R18 is 2-propenyl or 3-buten-1-ol when R18 is 3-butenyl, in the presence of a base, such as potassium carbonate, cesium carbonate or sodium hydride, preferably sodium hydride when R18 is 2-propenyl or cesium carbonate when R18 is 3-butenyl. The reaction is stirred in a polar solvent, such as dimethylformamide, at room temperature, for a time period between about 2 hours to about 48 hours, preferably about 18 hours.
In reaction 2 of Scheme 2, the compound of formula VIII is converted to the carboxylic acid compound of formula IV, wherein n is 2. The compound of formula VIII, wherein R18 is 2-propenyl, is converted to the compound of formula IV, wherein n is 2, by reacting VIII with borane-dimethylsulfide complex, followed by immediate oxidation using chromium trioxide in aqueous acetic acid. The oxidative cleavage of terminal olefins to carboxylic acids can be achieved by several methods known in the art. The preferred method for the oxidative cleavage of the compound of formula VIII, wherein R18 is 3-butenyl, to obtain the carboxylic acid compound of formula IV is to react VIII with sodium periodate in the presence of a catalytic amount of ruthenium (III) chloride in a mixture of carbon tetrachloride, acetonitrile and water.
The compound of formula IV, wherein n is 2, is further reacted to provide the hydroxamic acid compound of formula I, wherein n is 2, according to the procedure described above in reactions 4, 5 and 6 of Scheme 1.
An alternative method for the synthesis of the hydroxamic acid compound of formula I, wherein n is 1 and R3 and R4 are both hydrogen, is shown in reaction 1 of Scheme 3, beginning with reacting iminoacetic acid or a metal or ammonium salt of iminoacetic acid of formula X with a functional derivative of an arylsulfonic acid compound, such as an arylsulfonyl chloride, at room temperature, in the presence of a suitable base, such as triethylamine, and a polar solvent such as tetrahydrofuran, dioxane, water or acetonitrile, preferably a mixture of dioxane and water, to give the corresponding dicarboxylic acid compound of formula XI.
In reaction 2 of Scheme 3, the dicarboxylic acid compound of formula XI is dehydrated to give a cyclic anhydride compound of formula XII. The formation of cyclic anhydrides by dehydration of dicarboxylic acids may be achieved by a variety of means. The preferred method for the dehydration of the dicarboxylic acid compound of formula XI to give a cyclic anhydride compound of formula XII is to treat XI with an excess of acetic anhydride at a temperature between about 25xc2x0 C. to about 80xc2x0 C., preferably about 60xc2x0 C. Excess acetic anhydride and acetic acid, a by-product of the reaction, are removed by evaporation under reduced pressure leaving the cyclic anhydride compound of formula XII.
In reaction 3 of Scheme 3, the cyclic anhydride compound of formula XII is reacted, at room temperature, with an amine, NR1R2, or a salt of the amine, such as the hydrochloride, in the presence of a base, such as triethylamine, to give the carboxylic acid of formula II, wherein n is 1 and R3 and R4 are both hydrogen. Suitable solvents for the reaction are those that will not react with the starting materials, which include chloroform, methylene chloride and dimethylformamide, preferably methylene chloride.
The compound of formula II is further reacted to give the hydroxamic acid compound of formula I, wherein n is 1 and R3 and R4 are both hydrogen, according to the procedure described above in reaction 6 of Scheme 1.
In reaction 1 of Scheme 4, the carboxylic acid compound of formula IV, wherein n is 2, is converted to the corresponding compound of formula V, wherein R19 is (C1xe2x80x94C6)alkyl or tert-butyl, by reacting IV with a compound of the formula
(R19O)2CHN(CH3)2 
wherein R19 is (C1xe2x80x94C6)alkyl or tert-butyl, in an inert solvent, such as toluene, at a temperature between about 60xc2x0 C. to about 100xc2x0 C., for a time period between about 1 hour to about 3 hours, preferably 2hours. In reaction 2 of Scheme 4, the arylsulfonyl amino compound of formula VI wherein n is 1, 3, 4, 5 or 6 and R16 is (C1xe2x80x94C6)alkyl, benzyl, allyl or tert-butyl, is converted to the corresponding compound of formula XIII, wherein R19 is (C1xe2x80x94C8)alkyl or tert-butyl, by reacting VI with a reactive derivative of an alcohol of the formula 
such as the chloride, bromide or iodide derivative, preferably the bromide derivative, wherein R19 is (C1xe2x80x94C8)alkyl or tert-butyl, in the presence of a base such as potassium carbonate or sodium hydride, preferably sodium hydride, and a polar solvent, such as dimethylformamide. The reaction is stirred, at room temperature, for a time period between about 60 minutes to about 48 hours, preferably about 18 hours. The R16 protecting group, of the compounds of formulas IV and VI, is chosen such that it may be selectively removed in the presence of and without loss of the R19 (C1xe2x80x94C6)alkyl or tert-butyl group, therefore, R16 cannot be the same as R19. Removal of the R16 protecting group from the compound of formula XIII to give the corresponding carboxylic acid of formula XIV, wherein n is 1 to 6, in reaction 3 of Scheme 4, is carried out under conditions appropriate for that particular R16 protecting group in use which will not affect the R19 (C1xe2x80x94C6)alkyl or tert-butyl group,. Such conditions include; (a) saponification where R16 is (C1xe2x80x94C6)alkyl and R19 is tert-butyl, (b) hydrogenolysis where R18 is benzyl and R19 is tert-butyl or (C1xe2x80x94C6)alkyl, (c) treatment with a strong acid such as trifluoroacetic acid or hydrochloric acid where R16 is tert-butyl and R19 is (C1xe2x80x94C6)alkyl, or (d) treatment with tributyltinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride where R16 is allyl and R19 is (C1xe2x80x94C6)alkyl or tert-butyl.
In reaction 4 of Scheme 4, the carboxylic acid of formula XIV is converted to the to the hydroxamic acid compound of formula XV, wherein n is 1 to 6, by treating XIV with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenztriazole in a polar solvent, such as dimethylformamide, followed by the addition of hydroxylamine to the reaction mixture after a time period between about 15 minutes to about 1 hour, preferably about 30 minutes. The hydroxylamine is preferably generated in situ from a salt form, such as hydroxylamine hydrochloride, in the presence of a base, such as N-methylmorpholine. Alternatively, a protected derivative of hydroxylamine or its salt form, where the hydroxyl group is protected as a tert-butyl, benzyl or allyl ether, may be used in the presence of (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate and a base, such as N-methylmorpholine. Removal of the hydroxylamine protecting groups is carried out by hydrogenolysis for a benzyl protecting group or treatment with a strong acid, such as trifluoroacetic acid, for a tert-butyl protecting group. The allyl protecting group may be removed by treatment with tributyltinhydride and acetic acid in the presence of catalytic bis(triphenylphosphine) palladium (II) chloride. N,O-bis(4-methoxybenzyl)hydroxylamine may also be used, when R19 is (C1xe2x80x94C6)alkyl, as the protected hydroxylamine derivative where deprotection is achieved using a mixture of methanesulfonic acid and trifluoroacetic acid.
In reaction 5 of Scheme 4, the amide formula of formula XV is, if desired, converted to the corresponding carboxylic acid compound of formula XVI by (a) saponification where R19 is lower alkyl or (b) treatment with a strong acid, such as trifluoroacetic acid or hydrochloric acid, where R19 is tert-butyl.
Pharmaceutically acceptable salts of the acid compounds of the invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymetyl)-methylammonium slats.
Similarly acid addition salts, such as of mineral acids, organic carboxylic and organic sulfonic acids e.g. hydrochloric acid, methanesulfonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure
The ability of the compounds of formula I or their pharmaceutically acceptable salts (hereinafter also referred to as the compounds of the present invention) to inhibit matrix metalloproteinases or the production of tumor necrosis factor (TNF) and, consequently, demonstrate their effectiveness for treating diseases characterized by matrix metalloproteinase or the production of tumor necrosis factor is shown by the following in vitro assay tests.
Human recombinant collagenase is activated with trypsin using the following ratio: 10 xcexcg trypsin per 100 xcexcg of collagenase. The trypsin and collagenase are incubated at room temperature for 10 minutes then a five fold excess (50 xcexcg/10 xcexcg trypsin) of soybean trypsin inhibitor is added.
10 mM stock solutions of inhibitors are made up in dimethyl sulfoxide and then diluted using the following Scheme:
10 mMxe2x86x92120 xcexcMxe2x86x9212 xcexcMxe2x86x921.2 xcexcMxe2x86x920.12 xcexcM 
Twenty-five microliters of each concentration is then added in triplicate to appropriate wells of a 96 well microfluor plate. The final concentration of inhibitor will be a 1:4 dilution after addition of enzyme and substrate. Positive controls (enzyme, no inhibitor) are set up in wells D1-D6 and blanks (no enzyme, no inhibitors) are set in wells D7-D12.
Collagenase is diluted to 400 ng/ml and 25 xcexcl is then added to appropriate wells of the microfluor plate. Final concentration of collagenase in the assay is 100 ng/ml.
Substrate (DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2) is made as a 5 mM stock in dimethyl sulfoxide and then diluted to 20 xcexcM in assay buffer. The assay is initiated by the addition of 50 xcexcl substrate per well of the microfluor plate to give a final concentration of 10 xcexcM.
Fluorescence readings (360 nM excitation, 460 nm emission) were taken at time 0 and then at 20 minute intervals. The assay is conducted at room temperature with a typical assay time of 3 hours.
Fluorescence vs time is then plotted for both the blank and collagenase containing samples (data from triplicate determinations is averaged). A time point that provides a good signal (the blank) and that is on a linear part of the curve (usually around 120 minutes) is chosen to determine IC50 values. The zero time is used as a blank for each compound at each concentration and these values are subtracted from the 120 minutes data. Data is plotted as inhibitor concentration vs % control (inhibitor fluorescence divided by fluorescence of collagenase alonexc3x97100). IC50""s are determined from the concentration of inhibitor that gives a signal that is 50% of the control.
If IC50""s are reported to be  less than 0.03 xcexcM then the inhibitors are assayed at concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.03 xcexcM and 0.003 xcexcM.
Inhibition of gelatinase activity is assayed using the Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2 substrate (10 xcexcM) under the same conditions as inhibition of human collagenase (MMP-1).
72 kD gelatinase is activated with 1 mM APMA (p-aminophenyl mercuric acetate) for 15 hours at 4xc2x0 C. and is diluted to give a final concentration in the assay of 100 mg/ml. Inhibitors are diluted as for inhibition of human collagenase (MMP-1) to give final concentrations in the assay of 30 xcexcM, 3 xcexcM, 0.3 xcexcM and 0.03 xcexcM. Each concentration is done in triplicate.
Fluorescence readings (360 nm excitation, 460 emission) are taken at time zero and then at 20 minutes intervals for 4 hours.
IC50""s are determined as per inhibition of human collagenase (MMP-1) If IC50""s are reported to be less than 0.03 xcexcM, then the inhibitors are assayed at final concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.003 xcexcM and 0.003 xcexcM.
Inhibition of stromelysin activity is based on a modified spectrophotometric assay described by Weingarten and Feder (Weingarten, H. and Feder, J., Spectrophotometric Assay for Vertebrate Collagenase, Anal. Biochem. 147, 437-440 (1985)). Hydrolysis of the thio peptolide substrate [Ac-Pro-Leu-Gly-SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2H5] yields a mercaptan fragment that can be monitored in the presence of Ellman""s reagent.
Human recombinant prostromelysin is activated with trypsin using a ratio of 1 xcexcl of a 10 mg/ml trypsin stock per 28 xcexcg of stromelysin. The trypsin and stromelysin are incubated at 37xc2x0 C. for 15 minutes followed by 10 xcexcl of 10 mg/ml soybean trypsin inhibitor for 10 minutes at 37xc2x0 C. for 10 minutes at 37xc2x0 C. to quench trypsin activity.
Assays are conducted in a total volume of 250 xcexcl of assay buffer (200 mM sodium chloride, 50 mM MES, and 10 mM calcium chloride, pH 6.0) in 96-well microliter plates. Activated stromelysin is diluted in assay buffer to 25 xcexcg/ml. Ellman""s reagent (3-Carboxy-4-nitrophenyl disulfide) is made as a 1M stock in dimethyl formamide and diluted to 5 mM in assay buffer with 50 xcexcl per well yielding at 1 mM final concentration.
10 mM stock solutions of inhibitors are made in dimethyl sulfoxide and diluted serially in assay buffer such that addition of 50 xcexcL to the appropriate wells yields final concentrations of 3 xcexcM, 0.3 xcexcM, 0.003 xcexcM, and 0.0003 xcexcM. All conditions are completed in triplicate.
A 300 mM dimethyl sulfoxide stock solution of the peptide substrate is diluted to 15 mM in assay buffer and the assay is initiated by addition of 50 xcexcl to each well to give a final concentration of 3 mM substrate. Blanks consist of the peptide substrate and Ellman""s reagent without the enzyme. Product formation was monitored at 405 nm with a Molecular Devices UVmax plate reader.
IC50 values were determined in the same manner as for collagenase.
Human recombinant MMP-13 is activated with 2 mM APMA (p-aminophenyl mercuric acetate) for 1.5 hours, at 37xc2x0 C. and is diluted to 400 mg/ml in assay buffer (50 mM Tris, pH 7.5, 200 mM sodium chloride, 5 mM calcium chloride, 20 xcexcM zinc chloride, 0.02% brij). Twenty-five microliters of diluted enzyme is added per well of a 96 well microfluor plate. The enzyme is then diluted in a 1:4 ratio in the assay by the addition of inhibitor and substrate to give a final concentration in the assay of 100 mg/ml.
10 mM stock solutions of inhibitors are made up in dimethyl sulfoxide and then diluted in assay buffer as per the inhibitor dilution scheme for inhibition of human collagenase (MMP-1): Twenty-five microliters of each concentration is added in triplicate to the microfluor plate. The final concentrations in the assay are 30 xcexcM, 3 xcexcM, 0.3 xcexcM, and 0.03 xcexcM.
Substrate (Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2) is prepared as for inhibition of human collagenase (MMP-1) and 50 xcexcl is added to each well to give a final assay concentration of 10 xcexcM. Fluorescence readings (360 nM excitation; 450 emission) are taken at time 0 and every 5 minutes for 1 hour.
Positive controls consist of enzyme and substrate with no inhibitor and blanks consist of substrate only.
IC50""s are determined as per inhibition of human collagenase (MMP-1). If IC50""s are reported to be less than 0.03 xcexcM, inhibitors are then assayed at final concentrations of 0.3 xcexcM, 0.03 xcexcM, 0.003 xcexcM and 0.0003 xcexcM.
The ability of the compounds or the pharmaceutically acceptable salts thereof to inhibit the production of TNF and, consequently, demonstrate their effectiveness for treating diseases involving the production of TNF is shown by the following in vitro assay:
Human mononuclear cells were isolated from anti-coagulated human blood using a one-step FicoII-hypaque separation technique. (2) The mononuclear cells were washed three times in Hanks balanced salt solution (HBSS) with divalent cations and resuspended to a density of 2xc3x97105/ml in HBSS containing 1% BSA. Differential counts determined using the Abbott Cell Dyn 3500 analyzer indicated that monocytes ranged from 17 to 24% of the total cells in these preparations.
180xcexc of the cell suspension was aliquoted into flate bottom 96 well plates (Costar). Additions of compounds and LPS (100 ng/ml final concentration) gave a final volume of 200 xcexcl. All conditions were performed in triplicate. After a four hour incubation at 37xc2x0 C. in an humidified CO2 incubator, plates were removed and centrifuged (10 minutes at approximately 250xc3x97g) and the supernatants removed and assayed for TNFxcex1 using the RandD ELISA Kit.
For administration to humans for the inhibition of matrix metalloproteinases or the production of tumor necrosis factor (TNF), a variety of conventional routes may be used including orally, parenterally and typically. In general, the active compound will be administered orally or parenterally at dosages between about 0.1 and 25 mg/kg body weight of the subject to be treated per day, preferably from about 0.3 to 5 mg/kg. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The compounds of the present invention can be administered in a wide variety of different dosage forms, in general, the therapeutically effective compounds of this invention are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight.
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like a polyvinylpyrrolidone, sucrose, gelation and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulisfying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For parenteral administration (intramuscular, intraperitoneal, subcutaneous and intravenous use) a sterile injectable solution of the active ingredient is usually prepared. Solutions of a therapeutic compound of the present invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably adjusted and buffered, preferably at a pH of greater than 8, if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable intravenous injection purposes. The oil solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
The present invention is illustrated by the following examples, but it is not limited to the details thereof.